This study investigated the effects of acidity and metal ion on the antibacterial activity of alpha- and beta-chitosan at different molecular weights (Mw, 22-360 kDa) against Escherichia coli and Listeria innocua through agar well diffusion assay. Spectrophotometric, electrophoretic, and confocal fluorescence microscopy analysis were further employed to evaluate the antibacterial mechanisms probably involved. Increasing pH from 4.0 to 5.0 weakened the antibacterial ability of chitosan as shown by the decreased bacteria growth inhibition zone (BGIZ) from 0.63 to 0.57 cm for beta-chitosan (61 kDa) and from 0.62 to 0.57 cm for alpha-chitosan (30 kDa) against E. coli. All beta- and alpha-chitosan samples showed antibacterial activity against L. innocua, in which 22 kDa beta-chitosan and 30 kDa alpha-chitosan at pH 4.0 had the highest antibacterial activity with BGIZ of 1.22 and 0.98 cm, respectively. Interactive effect between pH and Mw on the antibacterial activity of beta-chitosan was observed, but not of alpha-chitosan. Adding Co2+ and Ni2+ significantly improved the antibacterial activity of chitosan, while adding K+, Na+, and Li+ significantly weakened the antibacterial activity of some beta- and alpha-chitosan samples (P < 0.05), and different Mw and forms of chitosan showed different metal ion absorption capacities. Results indicate that chitosan might insert into the groove of bacterial DNA double helix structure to induce DNA degradation and permeate through bacteria cell membranes and combine with genomic DNA to induce its dysfunction, providing evidences for the antibacterial mechanisms of chitosan.